PROJECT SUMMARY Hypoparathyroidism (HP) is a major toxicity of surgery for thyroid malignancy. Parathyroid (PT) glands are endocrine organs critical for maintaining calcium homeostasis. The most common cause of HP is iatrogenic - occurring after removal of the PT glands during dissection of the anterior and central neck compartment for the treatment of thyroid (or parathyroid) cancer. It is estimated that 5-7% of patients develop HP following thyroidectomy, and that the total number of adults with HP in the US is about 77,000. Some patients are unable to maintain stable physiological levels of Ca2+ and can suffer severe consequences including muscle cramping, tetany, and convulsions. The Krause laboratory is developing approaches for differentiation of autologous cells into functional PT cells. Identification and characterization of human PT stem/progenitor cells is critical for developing effective approaches for cell therapies for HP. The Krishnaswamy laboratory has developed a novel computational tool, PHATE (Potential of Heat-diffusion for Affinity-based Transition Embedding), which is able to capture intrinsic data geometry and denoise scRNA-seq data so that both local and global structures are apparent without imposing assumptions on the form of the data. PHATE is particularly powerful in analyzing differentiation data generated by dynamic processes where there is continuous variability between data points and thus is an ideal tool with which to dissect the cellular composition of complex tissues such as the PT gland. We hypothesize that the maintenance of the PT glands relies on stem/progenitor cell compartments that reside within supportive niches. We propose to define these cellular components and the maintenance mechanisms of PT tissue using scRNA-seq, PHATE, and functional studies through two specific aims. In the first, we will obtain primary PT glands and perform scRNA-Seq, which will then be analyzed with PHATE to reconstruct the cellular organization and the maintenance mechanisms of human PT gland. In the second, we will determine the functionality within the PT glands of specific cell populations determined by PHATE. Longterm in vivo function of PT cell subpopulations will be assessed by transplantation into immunodeficient mice. The studies proposed will shed light on the composition and cellular functions of different PT compartments, and will enhance our understanding of the homeostatic mechanisms that maintain the functional epithelial compartments of normal PT glands. The insight obtained will enhance understanding of the basic mechanisms of adult PT gland maintenance and regeneration, essential preclinical knowledge for the development of novel cellular therapies to restore PT function to patients with hypoparathyroidism.